The pulmonary manifestations of cystic fibrosis (CF) reflect a failure of lung defense leading to chronic airways bacterial infection. Several key aspects of the pathogenesis of early CF lung disease must be understood for best treatment approaches and biomarker development, including the observations that there is minimal CF lung disease at birth and that disease onset/progression is highly heterogeneous. Based on assumptions that 1) the CF lung exhibits a vulnerability to disease producing outside insults; 2) the vulnerability defect reflects localized loss of mucus clearance mechanisms due to airway surface dehydration, and 3) immobilized, relatively dehydrated mucus masses obstruct airways, stimulate inflammation, and serve as nidus for chronic bacterial infection, we hypothesize that the earliest CF lung disease will be characterized by quantifiable changes in airway mucin hydration/function, mucin stimulated macrophage activation, and airway hypoxia. This proposal will test the hypothesis in a unique CF cohort identified by newborn screening and studied at regular intervals with CT scans and bronchoscopy. Using samples obtained in the successful Australian AREST-CF program, we will develop a triad of novel measurement panels at U. North Carolina including: 1) biochemical and biophysical measurements of mucus hydration/function; 2) novel markers of inflammation, focusing on macrophages and 3) microbiome and metabolomic analysis of the spectrum of bacteria and their metabolic environment. We will determine whether these panels can quantify the burden of CF lung disease as defined by chest CT cross- sectionally and longitudinally. In parallel, we will develop minimally invasive, exhaled breath condensate based methods to assess these biomarkers. Successful testing of these hypotheses will generate both novel insights into the pathogenesis of CF lung disease and novel biomarkers to follow disease progression/therapeutic interventions. (End of Abstract)